Diketo-io



United States Patent F AlVHDES OF 9,12-DIKETOSTEARIC ACID; 9,12-DIKETO-10,11-OCTADECENOIC ACID; 9,12- DIKETO-10,11-D]HYDROXYSTEARICACID; AND 9,12 DIKETO 10,11 EPOXYSTEARIC ACID Joseph Nichols, Princeton,and Edgar S. Schipper, New

Brunswick, N. 1., assignors to Ethicon, Inc., a corporation of NewJersey No Drawing. Application May 5, 1955 Serial No. 506,353

7 Claims. or. 260348) This invention relates to amides of9,12-diketostearic acid, 9,12-diketo-10,ll-octadecenoic acid;9,12-diketo-10, ll-dihydroxystearic acid; and9,l2-diketo-10,11-epoxystearic acid, and more particularly relates toN-dialkylaminoalkyl-9,12-diketostearic acid amides;N-dialkylaminoalkyl-9,12-diketo-10,1l-octadecenoic acid amides; N-dialkylaminoalkyl 9,l2-diketo-10,1l-dihydroxystearic acid amides; and Ndialkylaminoalkyl 9,12 diketo 10,11- epoxystearic acid amides.

U. S. Patent No. 2,623,889, December 30, 1952, discloses the oxidationof l2-ketooleic acid and 12-ketoelaidic acid and esters of the acidswith chromic acid to produce a diketooctadecenoic acid, having anempirical formula of C H O and a melting point of 112 C. to 113 C. Theoxidation was carried out by means of excess chromic acid over thatrequired to oxidize a methylene group in the molecule, approximately 400percent excess being preferred for the best yields. The solvent mediumwas glacial acetic acid in an amount in excess of that required for asingle phase reaction medium and excess free sulfuric acid was alsopresent in an amount of at least about three times as much as requiredto form chromic acid from the soluble dichromate present in the reactionmixture. It was originally believed that the carbon-carbon double bondin the diketo-octadecencic acid prepared by the oxidation was in the9,10 position, but it has been since shown that the position of thecarbon double bond is 10,11.

9,12-diketo-10,ll-epoxystearic acid and 9,12-diketo-10,ll-dihydroxystearic acid may be prepared by the oxidation of9,l2-dil;eto-10,1l-octadecenoic acid with hydrogen peroxide. Oxidationwith hydrogen peroxide leading to the production of9,12-diketo-10,1l-epoxystearic acid is conducted in an alkaline mediumat a low temperature and is preferably conducted at a temperature notabove 5 C. in the presence of magnesium chloride which acts as acatalyst. Oxidation with hydrogen peroxide leading to the production of9,12-diketo-10,1l-dihydroxystearic acid is conducted in a neutral orslightly acidic medium and preferably at a temperature not exceeding 70C. in the presence of a catalytic amount of osmium tetroxide.

The novel N-dialkylaminoalkyl-9,IZ-diketostearic acid amides; Ndialkylaminoalkyl 9,12-diketo-10,11-octadecenoic acid amides,N-dialkylaminoalkyl-9,12-diketo-10, ll-dihydroxystearic acid amides, andN-dialkylamino-9, 12-diketo-10,1l-epoxystearic acid amides of thisinvention may be prepared by reacting equi-molar amounts of the acid anda lower alkyl chloroformate such as isobutylchloroformate,ethylchloroformate, or proplychloroformate 'in the presence of anequi-molar amount of a lower aliphatic tertiary amine, anacylalkylcarbonate be ing the result of the reaction. The reaction isconducted at a temperature below 0 C. and in the presence of an iceinert solvent such as tetrahydrofuran or toluene. The addition of anamine to the reaction mixture containing the acylalkylcarbonate resultsin the formation of the desired amide.

The novel amides of this invention have the following general structure:

in which Z is an ethylene, vinylene, epoxyethylene, or 1,2-dihydroxyethylene radical; X is a lower alkylene straight or branched-chainradical and preferably having two or three carbon atoms; and each of Rand R is a lower alkyl radical and preferably a methyl or ethyl radical.

For the purpose of illustration, the following examples are set forth toillustrate the preparation of the novel compounds of the invention butare not to be construed as limiting the spirit of the invention or itsscope.

EXAMPLE I N gamma-dimethylaminopropyl-QJ Z-diketo-J 0,1 1-

epoxystearamide 1.37 grams of isobutylchloroformate were added dropwiseto a stirred suspension of 3.26 grams of 9,12-diketo- 10,11-epoxystearicacid and 1.02 grams of triethylamine in solution in 200 ml. oftetrahydrofuran, the reaction mixture being maintained at 5 to 10 C.during the addition by means of an alcohol-Dry Ice bath. The reactionmixture was stirred and maintained at 5 C. to 10 C. for thirty minutesafter addition had been completed and 1.02 grams ofgamma-dimethylaminopropylamine were added. The reaction mixture wasrefluxed for thirty minutes at which time carbon dioxide was no longerevolved. The precipitate which formed was removed by ltration, thefiltrate was evaporated to dryness under reduced pressure and theresidue was suspended in 300 ml. of thrice normal hydrochloric acid. Thesuspension was decolorized with animal charcoal and filtered. Thefiltrate was neutralized with a concentrated aqueous solution ofpotassium hydroxide and the precipitate which formed was dried andrecrystallized from ethylacetate. 0.5 gram ofN-gamma-dimethylaminopropyl-9,IZ-diketo- 10,11-epoxystearamide having amelting point of 82- 83 C. were obtained.

Calculated for C H O N Percent Carbon 67.28

Hydrogen 10.31

Found:

Carbon 66.86 Hydrogen 10.50

EXAMPLE II N-beta-dimethylamin0ethyl-9,1Z-diketostearamia'e 2.74 gramsof isobutylchloroformate were added dropwise to a stirred solution of6.24 grams of 9,l2-diketostearic acid and 2.04 grams of triethylamine insolution in 200 ml. of tetrahydrofuran, the reaction mixture beingmaintained at 5 C. to 10 C. by means of an alcohol-Dry Ice bath. Thereaction mixture was stirred and maintained at this temperature forthirty minutes after additionhad been completed and 1.76 grams ofdimethylaminoethylamine' wereadded. The reaction mixture was refluxedfor fifteen minutes, at which time carbon dioxide was no longer evolved.The precipitate of triethylamine hydrochloride which formed was removedby filtration'and the filtrate was evaporated to dryness under reducedpressure. After the residue had been dissolved in 400 ml. of thricenormal hydrochloric acid the solution was decolorized with animalcharcoal and neutralized with a-concentrated solution of potassiumhydroxide. The precipitate which formed was dried and recrystallizedseveral times from ether and 4.8 grams ofN-betadimethylaminoethyl-9,l2-diketo-stearamide having a melting pointof 8788 C. were obtained.

Calculated for C H O N Percent Carbon 69.06 Hydrogen 11.07

Found: r r

Carbon 69.43 Hydrogen 11.11

7 EXAMPLE HI N- gam ma-diethylamiriopropyl-QJZ-diketo-10,11-

' octadecenoam ia e 2.74 grams of isobutylchloroformate were addeddropwise to a stirred solution of 6.2 grams of 9 ,l2-diketo-10,11-octadecenoic acid and 2.04 grams of triethylamine 'in solution in200 ml. of tetrahydrofuran, the reaction mixture being maintained 'at 5C. to -10 C. by means of an alcohol Dry Ice bath. The reaction mixturewas stirred and maintained at this temperature for thirty minutes afterthe addition. had been completed and 1.6 grams'ofgamma-diethylaminoproplyamine were added. The reaction mixture wasrefluxed for fifteen minutes at which time carbon dioxide was no longerevolved. The precipitate of triethylamine hydrochloride which formed wasremoved'by filtration and the filtrate was evaporated to dryness underreduced pressure. After the residue had been dissolved in 300 m1. ofthrice normal hydrochloric acid the solution was decolorized with animalcharcoal and the mixture was neutralized with aconcentrated solution ofpotassium hydroxide. The precipitate which formed was dried andrecrystallized consecutively from ether and ethyl acetate and 2.6 gramsof N gamma-diethy1aminopropyl-9,12-diketo-10,11-octa decenoamide havinga melting point of 9091 C. were 1.37 grams of isobutylchloroformate wereadded dropwise to a stirred solution of 3.44 grams of 9,12-diketo-10,11-dihydroxystearic acid and 1.02 grams of triethyl amine in solutionin 100 ml. of tetrahydrofuran, the reaction mixture being maintained at5 C. to 10 C. by means of an alcohol-Dry Ice bath. The reaction mixturewas stirred and maintained at this temperature for thirty minutes afterthe addition had been completed and a hot solution of 0.88 gram ofdimethylaminoethylamine in 50 ml. of tetrahydrofuran were added. Themixture was refluxed for thirty minutes at which time carbon dioxide wasno longer evolved. The precipitate of triethylamine hydrochloride whichformed was removed by filtration and the filtrate was evaporated todryness under reduced pressure. After the residue had been dissolved in300, m1. of thrice'normal hydrochloric acid the solution' wasd'colorized'with animal charcoal and the mixture was neutralized with aconcentrated solution of potassium hydroxide. The precipitate whichformed was dried and recrystallized several times from ether and 3.5grams of N-beta-dimethylaminoethyl-9,12-

diketo-10,11-dihydroxystearamide having a melting poin of 8284 C. wereobtained.

4.11 grams of isobutylchloroformate were'added dropwise to a stirredsuspension of 9.78 grams of 9,12-diketo- 10,11-epxystearic acid and3106' grams of triethylamine in solution in 500 ml. of tetrahydrofuran,the reaction mixture being maintained at C. to C. by

1 means of an alcohol-Dry Ice bath. The reaction mixture was stirred andmaintained at 5 to 10 C(for thirty minutes after the addition had beencompleted," and 3.48 grams of beta-diethylaminoethylamine were added. atwhich time carbon dioxide Was no longer evolved. The precipitate oftriethylamine hydrochloride which formed was removed by filtration andthe filtrate was evaporated to dryness under reduced pressure. Theresidue was dissolved in one liter of thrice'normal hydrochloric acidand. the. solution was treated with animal charcoal. The solution wasfiltered and made basic with potassium hydroxide and the precipitatewhich formed was recrystallized from ether. 4.4 grams ofN-betadiethylaminoethyl 9,12 diketo-10,1l epoxystearamide having amelting point of 84-85 C. were obtained.

obtained. 7

Calculated for C H O N Percent 'Carbon 71.04 Hydrogen 10.97

Found:

Carbon -eQ. 71.43

Hydrogen 10.96

EXAMPLE IV N-betq-dimethy lam in0'ethyl-9,12 diket0-1 0,1 Idihydroxystearamide diketo-10,1l-octadecenoamidehaving a melting pointof Hydrogen EXAMPLE VI N-b etadiethylaminoethyl-9,12-dikt0 Q 10,11octadeceno- I amide 4.11 grams of isobutylchloroformate were addeddropwise to a stirred solution of 9.3 grams of 9,12-diketo-l0,-

ll-octadecenoic acid. and 3.06 grams oftriethylaminein solution in 300ml. oftetrahydrofuran, the reaction mixture being maintained at 5 to -10C. by means of an alcohol-Dry'Ice bath. The mixture was stirred andmaintained at 5 to -10 C. for thirty minutes after the addition had beencompleted and 3.48 grams of beta-- diethylaminoethylamine were added.The mixture waspressure. The residue was dissolved in 500 ml. of thricenormal hydrochloric acidand the solution was decolorized with animalcharcoal, and neutralized with a concentratedsolution of potassiumhydroxide. The precipi tate which formed was dried and recrystallizedfrom ether, and 6.3"g'rams of N-beta-diethylaminoethyl-9,12-

96 C. were obtained.

Hydrogen 10.72"

The mixture was refluxed for fifteen minutes -5 EXAMPLE v11N-beta-diethylaminoethyl-9,12-diketstearamide 1.37 grams ofisobutylchloroformate were added dropwise to a stirred solution of 3.12grams of 9,12-diketostearic acid and 1.02 grams of triethylamine insolution in 100 ml. of tetrahydrofuran, the reaction mixture beingmaintained at to C. by means of an alcohol- Dry Ice bath. The mixturewas stirred and maintained at 5 to l0 C. for thirty minutes after theaddition had been completed and 1.16 grams ofbeta-diethylaminoethylamine were added. The reaction mixture wasrefluxed for fifteen minutes at which time carbon dioxide was no longerevolved. The precipitate of triethylamine hydrochloride which formed wasremoved by filtration and the filtrate was evaporated to dryness underreduced pressure. The residue was dissolved in 200 ml. of thrice normalhydrochloric acid and the solution was decolorized with animal charcoaland neutralized with a concentrated solution of potassium hydroxide. Theprecipitate which formed was dried, then dissolved in 20 ml. of ether,and the ether solution was cooled to C. 1.5 grams ofN-beta-diethylaminoethyl-9,12-diketostearamide having a melting point of8889 C. were obtained.

EXAMPLE VIII N-beta-diethylaminoethyl-9,12-a'iket0-10,1 1-

dihydroxyslearamide 4.11-grams of isobutylchloroformate were addeddropwise to a stirred solution of 10.32 grams of 9,12-diketo-10,11-dihydroxystearic acid and 3.06 grams of triethylamine in solutionin 300 ml. of tetrahydrofuran, the reaction mixture being maintained at5 C. to -10 C. by means of an alcohol-Dry Ice bath. The mixture wasstirred and maintained at 5 C. to -10 C. for thirty minutes after theaddition had been completed and 3.48 grams ofbeta-diethylaminoethylamine were added. The mixture was refluxed forfifteen minutes at which time carbon dioxide was no longer evolved. Theprecipitate of triethylamine hydrochloride which formed was removed byfiltration and the filtrate was evaporated to dryness under reducedpressure. The residue was dissolved in 500 ml. of thrice normalhydrochloric acid and the solution was decolorized with animal charcoal,filtered, and then neutralized with a concentrated solution of potassiumhydroxide. The precipitate which formed was dried and recrystallizedtwice from ether. 6.8 grams of N-beta-diethylaminoethyl-9,12-diketo10,11 dihydroxystearamide having a melting point of 7274 C. wereobtained.

The novel compounds of this invention are highly effective at lowconcentration in killing microorganisms or preventing or inhibitingtheir growth.

EXAMPLE IX N-beta-dimethylaminoethyl-9,12-diketostearamide, N- gammadiethylaminopropyl 9,12 diketo 10,11 octadecenoamide, N betadiethylaminoethyl 9,12 diketo- 10,11-epoxystearamide, N betadiethylaminoethyl-9,12-

9,12-diketo-10,1l-dihydroxystearamide were tested for bactericidalactivity by the following serial dilution method:

The compounds were sterilized by exposure to propylene oxide for threedays and 0.5 milliliter of sterile aqueous solution containing twentymicrograms of compound per milliliter of solution was added to 9.5milliliters of sterile yeast beef broth, the broth then being seriallydiluted with additional sterile broth to provide solutions of fivemilliliters total volume containing 500, 200, 100, 50, 10, 1, 0.1, and0.01 micrograms of compound per milliliter of solution. Four tubes, eachcontaining 4.5 milliliters of sterile broth, were inoculated with 0.1milliliter of a mature broth culture of Bacillus subtilis, Diplococcuspneumoniae III and Micrococcus pyogenes var. aureous respectively, andthe inoculated tubes were incubated at 37 C. for 24 hours. Four tubes,each containing 4.5 milliliters of sterile broth were each inoculatedwith 0.1 milliliter of an incubated culture and incubated at 37 C. for24 hours. Progressive series of dilutions ranging from 1 to 100, to 1 to1 billion, were prepared by dilution of the contents of the four tubeswith sterile broth and 0.1 milliliter of each dilution was transferredinto 4.5 milliliters of sterile broth and incubated at 37 C. for 24hours. 0.1 milliliter of the contents of the tubes representing thehighest dilution which initiated growth of the organisms were eachtransferred into each of the tubes containing the compounds to be testedand this was followed by incubation of the tubes at 37 C. for 48 hours.The table below gives the results of the tests by serial dilution incolumn I, the values being the concentrations in micrograms permilliliter at which growth was inhibited.

N-beta-dimethylaminoethyl-9,12 diketostearamide, N- gammadiethylaminopropyl-9,12 diketo 10,11 octadecenoamide, N betadiethylaminoethyl 9,12 diketo- 10,1 1-epoxystearamide,-N-beta-diethylaminoethyl-9,12-diketo-10,l l-octadecenoamide, Nbeta-diethylaminoethyl- 9,12 diketostearamide, and N betadiethylaminoethyl- 9,12-diketo-10,1l-dihydroxystearam'ide were testedfor activity against Mycobacteria tuberculosis H37Rv according to themethod of A. W. Frisch and M. S. Tarshis, American Review ofTuberculosis, vol. 64, page 551 (1951). The table below gives theresults of the activity of the novel compounds against Mycobacteriatuberculosis H37Rv in column II, inhibiting concentrations beingexpressed in micrograms per milliliter.

N-beta-dimethylaminoethyl-9,12 diketostearamide, N- gammadiethylaminopropyl 9,12 diketo 10,11 octadecenoamide, N betadiethylaminoethyl 9,12 diketo- 10,1 l-epoxystearamide,N-beta-diethylaminoethyl-9,12-diketo-10,1l-octadecenoamide, Nbeta-diethylaminoethyl- 9,12 diketostearamide, and N betadiethylaminoethyl- 9,l2-diketo-10,ll-dihydroxystearamide were tested foractivity against Coccidioides immitis by a serial dilution method givenby the following procedure:

The compounds were sterilized by exposure to propylene oxide for threedays and 0.25 milliliter of sterile aqueous solution containing twentymicrograms of compound per milliliter of solution was added to 4.5milliliters of sterile Mycophil broth, the broth then being seriallydiluted with additional sterile broth to provide solutions of fivemilliliters total volume containing 500, 100, 10, 1, 0.1, and 0.01micrograms of compound per milliliter of solution. One milliliter of aseventy-two hour Mycophil broth culture of Cryptococcus neoformans wasadded to ninety-nine milliliters of sterile Mycophil broth and 0.2milliliter of diluted culture was added to each of the serial dilutionscontaining the test compound and the inoculated tubes were incubated at25 C. for five days. The Table below gives the results of the tests incolumn III, inhibiting concentrations being expressed in micrograms permilliliter:

TABLE Bacte'rlal'lnhlhlting actiylty Miebb'adte- Column I Column 11Column III 1 Fungal. rial inhlbinhibiting T lting conconcen- Tcentratlon tration- D. sub- D. gmeu- M.p1 oae- Mycobucte- Cryptotilismaniac ms var. ri'um tubcaucus III aureus crc'ulosis moformam H37RvN-beta-dimethyl-aminoethyl-9,12-diketostearam1de 100 100N-g'amma-dlethyl-aminopropyLQR-dlketo-10,11-octade cenoamide 10 10 10100 10 N beta-diethyhaminoethyl-Q,l2ediketo-l0,ll-epoxystearamide 100 5050 25 500 N -beta-d iethyl-aminoethy1-9,12-diketo10,11-octadecen0am ldeI 10 10 10 10 N-beta-d.iethyl-aminoethyl-9,l2-dlketostearamide V 200 200200 100 500N-beta-diethyl-amlnoethyl-Q,12-diketo-10,1l-dihydroxVstearamide.. 500200 200 What is claimed is: 1. New and useful compounds-havingtheformula:

. 0 0 R l! H II C5H1;Q-Z-O(CHz)7-fO-NHX-N :5. N-- gammadiethylarninopropyl-9,12-diketo-10,11-

q fi q rloamide. g g V 'N beta y thyw,annexe-10,11-dihydioxystearamide.

7. N beta dietliylaminoethyl 9,12-diketo-10J1- epoxystearamide,

References Cited in the fileof this patent UNITED STATES PATENTS1,534,525 Hartmann et al. Apr. 21, 1925 2,430,004 De Groote et al. Nov.4, 1947 2,623,889 Nichols Dec. 30, 1952 2,626,876 Carnes Jan. 27, 19532,652,356 Hanson et al., Sept. 15, 1953 2,703,328 Celrner Mar. 1, 19.55

V .1, OTHER R F NC S h r Vaughan: J. Am. Chem. Soc., vol. 73 (1951),page 3547.

Emery et al.: J. Am. Chem. Soc. 1950), pages 1443- Squibb Abst.Bull:23': 7, page A 168, Feb. 15, 1950. a Journal of AmericanPharniJAssn, Science Ed., September' 1950, pages- 468489.

- Chein. Abst., vol. 40, 3795 1946;

1. NEW AND USEFUL COMPOUNDS HAVING THE FORMULA: